The discovery of RNA interference (RNAi) as a cellular mechanism that selectively degrades mRNAs allows for both the targeted manipulation of cellular phenotypes in cell culture and the potential for development of directed therapeutics (Behlke, Mol. Ther. 13, 644-670, 2006; Xie et al., Drug Discov. Today 11, 67-73, 2006).
Although siRNAs have great potential for manipulation of cellular phenotypes, due to their size and negative (anionic) charged nature, siRNAs are macromolecules with no ability to enter cells. Indeed, siRNAs are 25× in excess of Lipinski's “Rule of 5s” for cellular delivery of membrane diffusible molecules that generally limits size to less than 500 Da. Consequently, in the absence of a delivery vehicle or transfection agent, naked siRNAs do not enter cells, even at millimolar concentrations (Barquinero et al., Gene Ther. 11 Suppl 1, S3-9, 2004). Significant attention has been focused on the use of cationic lipids that both condense the siRNA and punch holes in the cellular membrane to solve the siRNA delivery problem. Although widely used, transfection reagents fail to achieve efficient delivery into many cell types, especially primary cells and hematopeotic cell lineages (T and B cells, macrophage). Moreover, lipofection reagents often result in varying degrees of cytotoxicity ranging from mild in tumor cells to high in primary cells.
Recent cell-directed targeting approaches of antibody fusions to DNA condensing protamine (Song et al., Nat. Biotechnol. 23, 709-717, 2005) and siRNA fusions to receptor targeted RNA aptamers (McNamara et al., Nat. Biotechnol. 24, 1005-1015, 2006) offer the potential to delivery siRNAs into select cells. While both approaches are promising, they fail to deliver siRNAs into 100% of tumor cells expressing the receptor, are not easily amendable to other non-receptor expressing cells, and have only been tested on a couple of cell types. Lastly, induction of aggregates to form nanoparticles by inclusion of cholesterol to form LDL particles and PEI condensation approaches or siRNA encapsulation in liposomes to mask the negative charge have been shown to deliver siRNAs with varying degrees of success into some tumor cells (Scherr et al., Ann. Hematol. 83, 1-8, 2004; Schiffelers et al., Nucleic Acids Res. 32, e149, 2004; Song et al., 2005; Soutschek et al., Nature 432, 173-178, 2004; Urban-Klein et al., Gene Ther. 12, 461-466, 2005; Zhang et al., Genet. Vaccines Ther. 3, 5, 2005). Thus, devising an approach to solve the siRNA macromolecular delivery problem that targets ˜100% of all cell types, primary and tumorigenic, by a rapid, non-cytotoxic mechanism remains important for expansion of RNAi potential in cell culture, target screening and therapeutic development.